Ca2+ is known to undergo transient alterations of intracellular concentration in nerve and other cells in response to diverse stimuli and to exert many of its actions by binding to a protein termed calmodulin (CaM). CaM, complexed with Ca2+, alters the activity of many cellular proteins including several protein kinases and a protein phosphatase which has been termed calcineurin (CN). Recently, this phosphatase has been implicated the regulation of exocytotic secretion in Paramecium tetraurelia. Our laboratory has produced several populations of monospecific, affinity-purified, domain-directed antibodies to the bovine brain CaM-dependent phosphatase. Two populations inhibit the enzyme by different mechanisms, while a third population produces a persistently activated, divalent cation independent phosphatase. In this application we propose to examine the effect of microinjecting these antibodies to the CaM-dependent phosphatase on Ca2+-induced exocytotic secretion and protein dephosphorylation in intact cultured adrenal chromaffin and PC-12 cells. Further, we plan to prepare adrenal chromaffin and PC-12 cells permeabilized with streptolysin O or digitonin. These cells are known to retain Ca2+-dependent exocytotic secretion of catecholamines while allowing the exchange of macromolecules with the "cellular" interior. The effect of our antibody populations on catecholamine secretion and protein dephosphorylation will be examined in these systems. The endogenase substrates of the CaM-dependent phosphatase will be identified in bovine adrenal medulla and purified as thiophosphorylated analogs by affinity chromatography on the immobilized, homogeneous phosphatase. The CaM- dependent phosphatase has been shown to exist as resolvable charge isoforms. The isoforms will be resolved by preparative, native isoelectric focussing and the chemical basis for micro-heterogeneity established with particular attention to examining the hypothesis that the different isoforms reflect different degrees of enzyme phosphorylation. The phosphorylation of the phosphatase in intact PC-12 cells in vitro by purified protein kinases will be studied. Finally, one of our populations of antibodies has been found to immuno precipitate a phosphoprotein phosphatase from a preparation of the CaM binding proteins of the filamentous fungus Aspergillus nidulans. We propose to purify this phosphatase by immunoaffinity chromatography and to compare its physical, kinetic and chemical properties with the bovine brain homolog.